Our goal is to define mitochondrial iron metabolism in eukaryotes at the molecular and biochemical level by studying yeast genes that are involved in mitochondrial iron transport and utilization. The information gained from yeast will be applied to the analysis of mammalian mitochondrial iron metabolism. The products of mitochondrial iron consuming processes, iron-sulfur cluster and heme synthesis, regulate cellular iron acquisition and storage. We have identified two cytosolic yeast proteins that regulate transcription of iron transport genes in response to the mitochondrial production of iron-sulfur clusters. We will determine how these proteins and iron-sulfur clusters regulate the transcription factor Aft1p. These proteins have vertebrate homologues and we will determine if the mammalian homologues are involved in cellular iron homeostasis. Our studies have shown that there are multiple mitochondrial iron transporters in yeast. Our genetic screens are designed to identify those transporters. At least one set of yeast mitochondrial iron transporters (MRS3/MRS4) has mammalian homologues (mitoferrin A and mitoferrin B). Using RNAi silencing reagents we will determine the contribution of these transporters to mitochondrial iron metabolism in specific cell types. Our data indicate that under normal conditions the size of the mitochondrial iron pool in both yeast and mammalian cells is tightly regulated. We will determine if mitochondrial iron import is regulated and if so what is the mechanism of regulation. We will utilize genetically engineered yeast with specific gene deletions and which express mammalian proteins such as mitochondrial ferritin, E. Coli iron-superoxide dismutase targeted to the mitochondria and human globin chains in the cytosol. We will determine if mitochondrial iron transporter number or activity is regulated and if iron efflux from the mitochondrial pool can also be regulated. We will determine how iron-consuming processes such as hemoglobin synthesis affect the flux of iron through the mitochondrial iron pool. Disruption in mitochondrial iron metabolism in yeast affects other cellular processes including activation of vacuolar iron transport and sterol synthesis. Using genetic and biochemical approaches, we will identify the signaling pathway by which decreased activity of mitochondrial iron transporters leads to activation of vacuolar iron import and how expression of mitochondrial transporters belonging to the cation diffusion facilitator family affect sterol biosynthesis.